Mechanical loading of bone initiates an anti-catabolic and anabolic gene response that promotes formation of a structurally competent skeleton. The work proposed in this competitive renewal will advance our study of the loaded response of the skeleton by examining a novel temporal sequence of gene regulation and deciphering whether orchestration of this anabolic process arises through a single initiating signal cascade. Our data reveal that mechanical strain regulates an early cluster consisting of canonical Wnt responders followed by a late cluster of anabolic genes, represented by Runx2, osterix and eNOS. This pattern of strain response is mirrored by gene response to shear force suggesting that we are studying a unified biomechanical response. A common signaling pathway involving HRas/ ERK1/2 is hypothesized to regulate those genes comprising the clustered response. This will be studied in SA#1. Our data further suggests a temporal pattern to the loading response: the canonical [unreadable]-catenin target response is vigorous at 4 h but returns to basal levels by 18 h while alterations in Runx2 and osterix do not occur until 18 h after application of loading. By silencing caveolin-1, a structural lipid raft molecule, we accelerate the temporal response of the Runx2-osterix gene cluster to within 4 hours of initiating load. Our characterization of the skeletal phenotype of the caveolin-1 deficient mouse further suggests that caveolin-1 attenuates mechanical signals that promote osteoblast differentiation, indicating that this molecule regulates the speed and/or magnitude of the osteogenic signal by sequestering [unreadable]-catenin. Causal relationships between early and late gene responses to mechanical stimulation will be the subject of SA#2, which also measures the response to in vivo loading in a caveolin-1 null mouse. Finally, SA#3 will examine whether the distal control regions of the RANKL promoter are regulated by strain and if similar elements are involved in mechanical regulation of genes associated with mature osteoblast activity. These will include genes studied in previous aims, as well as novel mechanical responders found by microarray. The work proposed will utilize strain and shear force applied to both primary murine stromal cells and the CIMC4 osteoblast line. Gene silencing (siRNA), manipulation of [unreadable]-catenin and ERK1/2 signaling networks, and the caveolin-1 null mouse are critical to methods. Dr. Gross will apply load to mice in vivo, Dr. Jacobs will advise on oscillatory shear experiments, and Dr. Pike will advise studies on load regulation of the RANKL promoter. In summary, our laboratory is in position to bring novel insight into understanding mechanisms by which loading generates an anti-catabolic/anabolic gene response.